The complement system comprises more than 30 serum and cellular proteins and has important roles in innate and adaptive immunity (Walport, M., N Engl J Med.; 344(14):1058-66, 2001). Complement activation can occur via three major pathways: the classical, alternative, and lectin pathways. The classical pathway is typically triggered by binding of a complex of antigen and IgM or IgG antibody to complement component C1. Activated C1 cleaves components C4 and C2 to produce C4a and C4b, in addition to C2a and C2b. C4b and C2a combine to form the classical pathway (CP)C3 convertase, which cleaves C3 to form C3a and C3b. Binding of C3b to C3 convertase produces a C5 convertase. The alternative pathway (AP) is typically activated by targets such as microbial surfaces and various complex polysaccharides and other materials. This pathway can be initiated by spontaneous cleavage of the thioester bond in C3 to form C3(H2O). C3(H2O) binds factor B, which allows factor D to cleave factor B to Ba and Bb. Bb remains associated with C3(H2O) to form the C3(H2O)Bb complex, which acts as a C3 convertase and cleaves C3, resulting in C3a and C3b. C3b formed either via this process or via the classical or lectin pathways binds to targets, e.g., on cell surfaces, and forms a complex with factor B, which is later cleaved by factor D to form Bb, resulting in a C3 convertase. This alternative pathway convertase is also termed C3bBb. Binding of another molecule of C3b to the C3 convertase produces a C5 convertase. The lectin complement pathway is initiated by binding of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein localized in the intermediate region between the endoplasmic reticulum and the Golgi. The MBL-2 gene encodes the soluble mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in proteolysis of C4 and C2, leading to a C3 convertase, which leads to production of a C5 convertase as described above for the classical pathway.
C5 convertases generated via any of the pathways cleave C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzes polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The MAC inserts itself into target cell membranes and causes cell lysis. Sub-lytic amounts of MAC on the membrane of cells may affect cell function in a variety of ways. The small cleavage products C3a, C4a, and C5a are anaphylotoxins and mediate multiple reactions in the acute inflammatory response. C3a and C5a are also chemotactic factors that attract immune system cells such as neutrophils. All three anaphylatoxins contain carboxyl-terminal arginine residues that are rapidly removed by carboxypeptidases in whole blood or serum. The desarginine derivatives of C3a and C4a exhibit minimal binding to the C3a receptor. In the case of C5a, the biologic activity and decreases by more than an order of magnitude.
Deficiencies and functional disorders in complement components or regulatory proteins are responsible for a number of disorders. It is also increasingly recognized that excessive or inappropriate complement activation plays an important pathogenic role in a wide variety of diseases and clinical conditions. A variety of assays have been developed for studying the complement system, diagnosing complement deficiencies and complement-mediated disease, and developing agents to modulate the complement system. Hemolysis-based techniques may be used to measure the functional activity of the entire complement cascade. Classical or alternative complement pathway activation may be assessed by measuring complement-mediated hemolysis of erythrocytes (e.g., antibody-sensitized or unsensitized sheep or rabbit erythrocytes) by human serum or by a set of complement components. For example, to measure the functional capacity of the classical pathway, sheep red blood cells coated with hemolysin (rabbit IgG to sheep red blood cells) are typically used as target cells (sensitized cells). These Ag-Ab complexes activate the classical pathway and result in lysis of target cells when the complement components are functional and present in sufficient concentration. To determine the functional capacity of the alternative pathway, rabbit red blood cells are typically used as the target cell.
A variety of immunological techniques have been developed to measure the concentration of a particular complement component or cleavage product. Antibodies have been raised against different epitopes of various (human) complement components and their major cleavage products. Radioimmunoassays, radial diffusion assays, and enzyme-linked immunosorbent assays (ELISAs) have been developed to detect these molecules. Descriptions of various methods that have been employed in efforts to measure complement activation are found in Wurzer, R., “Immunochemical Measurement of Complement Components and Activation Products”, pp. 103-112, in Complement Methods and Protocols (Methods in Molecular Biology) Humana Press; 1 edition, 2000, in the review article by Mollnes, T., et al., “Complement analysis in the 21st century”, Mol. Immunol., 44: 3838-3849, 2007, and references therein, all of which are incorporated herein by reference.